17-DMAG regulates NF-κB activity and target gene transcription. (A) Western blot analysis for IκBα and phosphorylated IκBα (P-IκBα) in cytoplasmic cell lysate and p65 and p50 in nuclear cell lysates prepared from CLL patient cells treated with vehicle control or 1μM 17-DMAG for 24 hours, 20 ng/mL TNF-α for 30 minutes, or 10μM Bay-11 for 1 hour. Blots are probed with actin or BRG1 as a loading control. Results shown are representative of at least 6 patient samples. (B) Electrophoretic mobility shift assay analysis with nuclear extract prepared from cells treated with vehicle control or 1μM 17-DMAG for 24 hours, 20 ng/mL TNF-α for 30 minutes, or 10μM Bay-11 for 1 hour using a radiolabeled oligonucleotide containing a consensus NF-κB-binding site. Antibody shifts are performed with nuclear extract prepared from vehicle control sample incubated with antibodies specific to the p65 or p50 subunits of NF-κB. The p65/p50 and p50/p50 complexes are indicated. Results shown are representative of 6 CLL samples. (C) Real-time PCR for MCL1 and BCL2 (n = 5 and n = 11, respectively) after 24 hours 1μM 17-DMAG treatment. Data are normalized to 18S transcript and represented as fold change in expression of 17-DMAG treated relative to the vehicle control. ○ represents individual patient samples. Bar represents the average of all patient samples. (D) Western blot analysis for MCL1 and BCL2 in cytoplasmic cell lysate prepared from CLL patient cells grown treated with vehicle control or 1μM 17-DMAG for 24 hours. Blots are probed with actin as a loading control. Results shown are representative of at least 6 patient samples. (E) Real-time PCR for MCL1 (n = 6) in CLL patient cells treated with vehicle control or 1μM 17-DMAG for 24 hours in the presence or absence of 100μM caspase inhibitor z-VAD-fmk. Data are normalized to TBP or 18S transcript and represented as fold change in expression of 17-DMAG treated relative to the vehicle control. ○ represents individual patient samples. Bar represents the average of all patient samples.